SNP (Single Nucleotide Polymorphism) molecular marker linked with sclerotinia rot resistance QTL-C06 site of brassica napus and application of SNP molecular marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker linked with a sclerotinia rot resistance QTL-C06 site of brassica napus and application of the SNP molecular marker. The SNP molecular marker is located between the 8737061 basic group and the 9396018 basic group of the C06 chromosome of the brassica napus. The QTL locus disclosed by the invention is stable in expression and high in contribution rate in associated populations, and has a good breeding application prospect. The marker information and the detection method are simple and easy to implement, are suitable for the background of different brassica napus varieties, and have wide adaptability and popularization potential.

Description

Technical Field

[0001] The present invention relates to the technical fields of molecular biology and rapeseed breeding, and in particular to a SNP molecular marker linked to a QTL-C06 site of sclerotinia rot resistance in Brassica napus and applications thereof. Background Art

[0002] Rapeseed (Brassica napus) is one of the most important oilseed crops in the genus Brassica in the family Cruciferae. It is widely cultivated in many countries and regions around the world and is the only large-scale oilseed crop grown in my country during winter. It plays a strategic role in ensuring national edible oil supply security. Brassica napus, cultivated in my country, has become the primary cultivated variety due to its excellent agronomic traits, including strong disease resistance, high yield, wide adaptability, and good stress tolerance. Rapeseed yield increases primarily rely on three key factors: increasing yield per unit area, increasing seed oil content, and expanding cultivated areas. Improving yield and oil content are the core goals of current rapeseed breeding efforts. However, key traits such as yield, oil content, and quality are complex quantitative traits regulated by multiple genes and significantly susceptible to environmental influences. Therefore, traditional breeding methods have been unable to achieve breakthroughs in the short term. In recent years, molecular breeding techniques, particularly the integration of quantitative genetics with marker-assisted selection (MAS), have provided more efficient and precise approaches for rapeseed breeding.

[0003] Single nucleotide polymorphisms (SNPs) are third-generation molecular markers that are widely present in the genome. They manifest as single-base variations between individuals at the same gene locus, including base substitutions, insertions, or deletions. SNPs offer advantages such as high throughput, automation, and robustness, making them a crucial tool for quantitative trait gene mapping and marker-assisted breeding. They are particularly well-suited for genetic analysis of complex traits in polyploid plants such as rapeseed.

[0004] Sclerotinia sclerotiorum is one of the major diseases affecting rapeseed production. It is caused by the pathogenic fungus Sclerotinia sclerotiorum. This pathogen has a wide host range and can infect more than 600 plant species, posing a serious threat to rapeseed yield and quality. Currently, there are no rapeseed varieties that show high resistance to sclerotinia sclerotiorum, but there is a relatively rich resistance variation in natural germplasm resources. This resistance is a quantitative trait jointly regulated by multiple genes. With the completion of whole-genome sequencing of rapeseed and the rapid development of molecular marker technology, more and more quantitative trait loci (QTLs) related to sclerotinia sclerotiorum resistance have been preliminarily located. However, most existing studies remain at the preliminary positioning stage and lack in-depth analysis of the resistance mechanism.

[0005] Therefore, further QTL positioning and association analysis of the resistance to bacterial sclerotinia in Brassica napus based on efficient molecular marker technologies such as SNP will help to discover key disease-resistant genes, promote the process of molecular marker-assisted breeding, and provide theoretical support for clarifying its resistance genetic mechanism. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention proposes a single-nucleotide polymorphism (SNP) molecular marker linked to the QTL C06 locus for resistance to sclerotinia sclerotiorum in Brassica napus and its application. Through precise genome-wide association analysis, the present invention screens and applies SNP markers significantly associated with resistance to sclerotinia sclerotiorum in Brassica napus leaves, enabling early, stable, and efficient identification of resistant materials and molecular marker-assisted breeding.

[0007] The invention provides a SNP molecular marker of a QTL site for sclerotinia sclerotiorum resistance in Brassica napus. The SNP molecular marker is located between the 8737061st base and the 9396018th base of the C06 chromosome of Brassica napus.

[0008] In some embodiments, the SNP molecular marker is located at base 8737061 of chromosome C06 of Brassica napus, and the base 8737061 exhibits polymorphism of two alleles, T and G.

[0009] In some embodiments, the SNP molecular marker is located at base 9396018 of chromosome C06 of Brassica napus, and the base 9396018 exhibits polymorphism of two alleles, C and T.

[0010] In some embodiments, the SNP molecular marker is located at base 8847348 of chromosome C06 of Brassica napus, and the base 8847348 exhibits polymorphism of two alleles, T and A.

[0011] The present invention also provides the use of the SNP molecular marker in any of the following:

[0012] (1) Detect and / or predict resistance to sclerotinia sclerotiorum in Brassica napus;

[0013] (2) Selecting Brassica napus for its resistance to Sclerotinia sclerotiorum;

[0014] (3) Molecular marker-assisted breeding of Brassica napus;

[0015] (4) Accelerate the breeding process of Brassica napus varieties for resistance to Sclerotinia sclerotiorum.

[0016] The present invention also provides a primer or probe, comprising a primer or probe for amplifying or detecting the SNP molecular marker.

[0017] In some embodiments, the primers are designed based on any one of the following DNA fragments:

[0018] (1) DNA fragments containing 400 bp before and after base 8737061 of the C06 chromosome of Brassica napus;

[0019] (2) DNA fragments containing 400 bp before and after base 9396018 on the C06 chromosome of Brassica napus;

[0020] (3) DNA fragments with 400 bp sequence before and after base 8847348 of the Brassica napus C06 chromosome.

[0021] In some embodiments, (1) the nucleotide sequence of the DNA fragment is as shown in SEQ ID NO.1;

[0022] (2) The nucleotide sequence of the DNA fragment is shown in SEQ ID NO. 2;

[0023] (3) The nucleotide sequence of the DNA fragment is shown in SEQ ID NO.3.

[0024] In some embodiments, the primers are any one of the following groups:

[0025] (1) The nucleotide sequences shown in SEQ ID NOs. 4 to 5;

[0026] (2) the nucleotide sequences shown in SEQ ID NOs. 6 to 7;

[0027] (3) The nucleotide sequences shown in SEQ ID NOs. 8 to 9.

[0028] Preferably, the primer or probe is labeled with a fluorescent group, and the fluorescent group includes FAM, HEX, VIC, and ROX.

[0029] The present invention also provides a reagent or a kit comprising the primer or the probe.

[0030] The present invention also provides a rapeseed breeding method, comprising the following steps: extracting genomic DNA of the rapeseed to be tested, testing the SNP molecular markers, and screening plants with high resistance to sclerotinia sclerotinia according to the test results for breeding.

[0031] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0032] 1. The present invention located a QTL interval significantly associated with sclerotinia resistance on chromosome C06 of Brassica napus, defined between bases 8737061 and 9396018, with a contribution rate of 10.77% to the sclerotinia resistance phenotype, providing a clear target for subsequent functional gene mining and resistance breeding.

[0033] 2. Compared with the traditional disease nursery inoculation method, the SNP markers provided by the present invention can directly predict plant resistance in the early stage, without being affected by factors such as pathogen pressure and climatic conditions. The screening efficiency is high and the repeatability is strong, which greatly reduces the breeding cycle and cost.

[0034] 3. Using key SNP markers such as chrC06_8847348 to conduct molecular marker-assisted selection of germplasm materials can quickly identify disease-resistant genotypes, achieve precise breeding and elimination, and improve the efficiency and success rate of resistance breeding.

[0035] 4. The QTL loci of this invention are stable in the associated population, with a high contribution rate, and have good breeding application prospects. Its marker information and detection method are simple and easy to use, applicable to different Brassica napus varieties, and have broad adaptability and promotion potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 This is a frequency distribution diagram of the Sclerotinia disease resistance phenotype in the associated population of Example 1 of the present invention.

[0038] Figure 2 This is the Manhattan plot of the association analysis of the Sclerotinia disease resistance phenotype in the associated population of Example 3 of the present invention.

[0039] Figure 3 This is the molecular marker allelic analysis map of the peak SNP (chrC06_8847348) of the resistance QTL site in Example 3 of the present invention. DETAILED DESCRIPTION

[0040] To help those skilled in the art better understand the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0041] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are all commercially available.

[0042] Example 1 Evaluation of resistance to sclerotinia sclerotiorum in Brassica napus

[0043] (1) A natural population of pure lines of Brassica napus (Brassica napus) consisted of 220 accessions, 169 of which were from China and 51 from abroad (published in Zhang, Y., Structural variation reshapes population gene expression and trait variation in 2,105 Brassica napus accessions. Nature Genetics 2024, 56(11), 2538-2550.). A survey on leaf sclerotinia resistance was conducted at the Yangluo Experimental Base in Wuhan and the Yangzhou Academy of Agricultural Sciences Experimental Base.

[0044] (2) Direct seeding was used, with a row spacing of 33 cm and a plant spacing of 15 cm. Each plot had 4 rows and three replicates were randomly designed. Protective rows were planted around the test material plots.

[0045] (3) Evaluation of resistance to sclerotinia sclerotiorum: Mature leaves of 10 plants in each plot were infected with Sclerotinia sclerotiorum. The lesion area after inoculation was recorded, and the lesion expansion area between each two time points was calculated.

[0046] The disease resistance phenotypes of the 220 materials were summarized as follows:

[0047] Table 1 Sclerotinia disease resistance phenotypes of 220 accessions

[0048]

[0049]

[0050] The results of the leaf lesion expansion area distribution of the associated population showed that the phenotypic distribution of the lesion expansion area was normally distributed, such as Figure 1 shown.

[0051] Example 2 Acquisition of high-quality SNP datasets from associated populations

[0052] The CTAB method was used to extract total DNA from leaves. The specific method was as follows:

[0053] The young leaves were rinsed in 10% ethanol; 0.1-0.2 g of leaves were cut and placed in a mortar, quickly ground with liquid nitrogen to powder, and placed in a 2 mL centrifuge tube; 700 μL of preheated DNA extraction solution was added; after mixing, the tube was placed in a 65°C water bath for 1 hour, mixing once every 10-15 minutes; 700 μL of a mixture (phenol: chloroform: isoamyl alcohol = 25:24:1) was added and gently inverted to mix for 10 minutes; centrifuged at 10,000 × g for 15 minutes at room temperature; the supernatant was transferred to a new 2 mL centrifuge tube; an equal volume of a mixture (chloroform: isoamyl alcohol = 24:1) was added and inverted to mix, allowed to stand for 5 minutes, and centrifuged at 10,000 × g for 15 minutes. The supernatant was pipetted into a new centrifuge tube; 2 volumes of anhydrous ethanol were added, mixed, and allowed to stand at -20°C for 1 hour, 10 The pellet was centrifuged at 10,000 × g for 10 min, and the supernatant discarded. The pellet was then washed with 500 μL of pre-cooled 75% ethanol, and the supernatant discarded. After two consecutive washes, the pellet was air-dried. 100 μL of a 2% RNase A solution was added, and the pellet was incubated at 37°C for 1 h before incubating at 4°C overnight. DNA was extracted again with an equal volume of a mixture (chloroform:isoamyl alcohol = 24:1), mixed by inversion, and incubated for 10 min. The pellet was centrifuged at 10,000 × g for 15 or 20 min to remove RNase A. The supernatant (approximately 60 μL) was aspirated and centrifuged again for 1 min. DNA concentration, quality, and integrity were assessed by agarose gel electrophoresis (0.8%) and UV spectrophotometry. The absorbance 260 / 280 ratio for all DNA samples was determined to be between 1.8 and 2.0. DNA samples were then shipped on dry ice to a sequencing company (BGI Genomics Co., Ltd.), with each sample sequenced at a depth of approximately 7×.

[0054] After obtaining high-quality DNA as described above, the sequencing company (BGI Genomics) performed 7× coverage depth sequencing and returned data. FastQC software was used to assess sequencing quality, followed by adapter and low-quality read filtering. Clean data from paired-end sequencing of each material was obtained, followed by mapping using bwa software and variant detection using GATK software. After obtaining the total SNP dataset for the associated population, the SNP dataset was quality-filtered based on a minimum allele frequency ≥ 0.05, a missingness rate ≤ 0.1, and a heterozygosity rate ≤ 0.1. Ultimately, a high-quality population SNP dataset was obtained for subsequent analysis.

[0055] Example 3 Genome-wide association analysis

[0056] Based on the VCF file of the high-quality SNP dataset generated in step 2 of Example 2, genome-wide association analysis of sclerotinia-related phenotypes was performed using the rMVP package of R language using the GLM model to obtain the P value of each SNP site. When the P value was less than 2.98×10 -7 The SNP with the smallest P value is the peak SNP. The materials with different allele types of the peak SNP in the population are grouped and variance analysis is performed. The percentage of the ratio of the variance between groups to the total variance is the contribution rate of the peak SNP. Manhattan plot of association analysis of Sclerotinia disease resistance phenotype in the associated population is shown below. Figure 2 shown.

[0057] Through analysis, the interval of the QTL locus for Sclerotinia sclerotiorum resistance in Brassica napus was confined to bases 8737061 to 9396018 on chromosome C06 of Brassica napus. The corresponding SNPs were chrC06_8737061 (T / G) and chrC06_9396018 (C / T), and the peak SNP was chrC06_8847348 (T / A). The contribution rate of this QTL to Sclerotinia sclerotiorum resistance in Brassica napus was 10.77% (the materials were grouped according to the different allele types of the peak SNP, and a one-way analysis of variance was performed. The percentage of the variance between groups divided by the total variance was the contribution rate).

[0058] The peak SNP for Sclerotinia resistance is chrC06_8847348 (T / A), and the corresponding Sclerotinia resistance phenotype grouping is: when the SNP at the chrC06_8847348 position is T, the average lesion expansion area of ​​the material is 3.39; when it is A, the average lesion expansion area of ​​the material is 4.86; the contribution rate of this peak SNP is 10.77%; the molecular marker allelic analysis diagram of the peak SNP (chrC06_8847348) of the resistance QTL site is shown in Figure 3 shown.

[0059] One boundary SNP marker of the sclerotinia phenotype is: chrC06_8737061 (T / G), and the corresponding sclerotinia resistant phenotype grouping is: when the SNP at the chrC06_8737061 position is T, the average lesion expansion area of ​​the material is 3.40; when it is G, the average lesion expansion area of ​​the material is 4.64; the contribution rate of this boundary SNP is 8.4%; another boundary SNP marker of the sclerotinia phenotype is: chrC06_9396018 (C / T), and the corresponding sclerotinia phenotype grouping is: when the SNP at the chr C06_9396018 position is C, the average lesion expansion area of ​​the material is 3.38; when it is T, the average lesion expansion area of ​​the material is 4.62; the contribution rate of this boundary SNP is 9.16%.

[0060] The complete genome sequence of Brassica napus has been published, including the 400 bp sequence before and after chrC06_8737061(T / G) (801 bp in total) as shown in SEQ ID NO.1, the 400 bp sequence before and after chrC06_9396018(C / T) (801 bp in total) as shown in SEQ ID NO.2, and the 400 bp sequence before and after chrC06_8847348(T / A) (801 bp in total) as shown in SEQ ID NO.3. Those skilled in the art can use conventional methods to design specific primers or probes for detecting SNP sites based on known sequences. The primers or probes can also be labeled with fluorescent groups such as FAM, HEX, VIC, ROX, etc., and quenching groups such as BHQ1 or TAMRA through conventional techniques in the art, so that the genotype of the above-mentioned SNP site can be detected by conventional methods in the art such as sequencing or PCR. This can detect the level of resistance of Brassica napus to sclerotinia, predict the level of resistance of Brassica napus to sclerotinia, and then effectively select the level of resistance of Brassica napus to sclerotinia, which can be used for molecular marker-assisted breeding of Brassica napus with resistance to sclerotinia, thereby accelerating the process of breeding Brassica napus for resistance to sclerotinia.

[0061] Therefore, the present invention detected a QTL site for Sclerotinia sclerotinia resistance in Brassica napus on chromosome C06 through phenotypic analysis of leaf Sclerotinia sclerotinia resistance and whole-genome resequencing, followed by whole-genome association analysis, with a contribution rate of 10.77% to Sclerotinia sclerotinia resistance in Brassica napus. The QTL locus for sclerotinia sclerotinia resistance in Brassica napus is located between bases 8737061 and 9396018 on chromosome C06 of Brassica napus. The boundary significant SNPs are chrC06_8737061 (T / G) and C06_9396018 (C / T), and the peak SNP is chrC06_8847348 (T / A). The SNP molecular marker tightly linked to the QTL locus can be used to detect the level of sclerotinia sclerotinia resistance in Brassica napus leaves, predict the level of sclerotinia sclerotinia resistance in Brassica napus, effectively select Brassica napus for the level of sclerotinia sclerotinia resistance, and then be used for molecular marker-assisted breeding of Brassica napus for sclerotinia sclerotinia resistance, thereby accelerating the process of breeding Brassica napus for sclerotinia sclerotinia resistance.

[0062] The molecular marker-assisted selection is carried out by the SNP molecular markers disclosed by the present invention. The identification method is simple, the selection efficiency is high, and the resistance of Brassica napus to sclerotinia rot can be predicted. The selection target is clear and is not affected by the environment. Brassica napus single plants with sclerotinia rot resistance can be identified in the early growth stage of Brassica napus, and other single plants are eliminated. In summary, the QTL locus for sclerotinia rot resistance in Brassica napus leaves of the present invention has a high contribution rate to the resistance to sclerotinia rot of Brassica napus, plays a key role in the regulation of the resistance to sclerotinia rot of Brassica napus, can be used for map-based cloning and molecular marker-assisted selection, and is suitable for large-scale promotion and application.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0064]

[0065]

Claims

1. A SNP molecular marker for a QTL locus for resistance to sclerotinia sclerotiorum in Brassica napus, characterized in that: The SNP molecular marker is located between the 8737061st base and the 9396018th base of the C06 chromosome of Brassica napus.

2. The SNP molecular marker according to claim 1, characterized in that The SNP molecular marker is located at the 8737061st base of the C06 chromosome of Brassica napus, and the 8737061st base has two allele polymorphisms, T and G.

3. The SNP molecular marker according to claim 1, characterized in that The SNP molecular marker is located at the 9396018th base of the C06 chromosome of Brassica napus, and the 9396018th base shows polymorphism of two alleles, C and T.

4. The SNP molecular marker according to claim 1, characterized in that The SNP molecular marker is located at the 8847348th base of the C06 chromosome of Brassica napus, and the 8847348th base has two allele polymorphisms, T and A.

5. Use of the SNP molecular marker according to any one of claims 1 to 4 in any one of the following: (1) Detect and / or predict resistance to sclerotinia sclerotiorum in Brassica napus; (2) Selecting Brassica napus for its resistance to Sclerotinia sclerotiorum; (3) Molecular marker-assisted breeding of Brassica napus; (4) Accelerate the breeding process of Brassica napus varieties for resistance to Sclerotinia sclerotiorum.

6. A primer or probe, characterized in that The method comprises a primer or a probe for amplifying or detecting the SNP molecular marker according to any one of claims 1 to 4.

7. The primer or probe according to claim 6, characterized in that The primers are designed based on any one of the following DNA fragments: (1) DNA fragments containing 400 bp before and after base 8737061 of the C06 chromosome of Brassica napus; (2) DNA fragments containing 400 bp before and after base 9396018 on the C06 chromosome of Brassica napus; (3) DNA fragments with 400 bp sequence before and after base 8847348 of the Brassica napus C06 chromosome.

8. The primer or probe according to claim 7, characterized in that (1) The nucleotide sequence of the DNA fragment is shown in SEQ ID NO. 1; (2) The nucleotide sequence of the DNA fragment is shown in SEQ ID NO. 2; (3) The nucleotide sequence of the DNA fragment is shown in SEQ ID NO.

3.

9. A reagent or kit, characterized in that Comprising the primer or probe according to any one of claims 6 to 8.

10. A rapeseed breeding method, characterized in that: The following steps are involved: The genomic DNA of the rapeseed to be tested is extracted, the SNP molecular marker according to any one of claims 1 to 4 is tested, and plants with high resistance to sclerotinia sclerotiorum are screened out for breeding according to the test results.

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